Mesenchymal stem cell-macrophage crosstalk and bone healing

Abstract

Recent research has brought about a clear understanding that successful fracture healing is based on carefully coordinated cross-talk between inflammatory and bone forming cells. In particular, the key role that macrophages play in the recruitment and regulation of the differentiation of mesenchymal stem cells (MSCs) during bone regeneration has been brought to focus. Indeed, animal studies have comprehensively demonstrated that fractures do not heal without the direct involvement of macrophages. Yet the exact mechanisms by which macrophages contribute to bone regeneration remain to be elucidated. Macrophage–derived paracrine signaling molecules such as Oncostatin M, Prostaglandin E2 (PGE2), and Bone Morphogenetic Protein-2 (BMP2) have been shown to play critical roles; however the relative importance of inflammatory (M1) and tissue regenerative (M2) macrophages in guiding MSC differentiation along the osteogenic pathway remains poorly understood. In this review, we summarize the current understanding of the interaction of macrophages and MSCs during bone regeneration, with the emphasis on the role of macrophages in regulating bone formation. The potential implications of aging to this cellular cross-talk are reviewed. Emerging treatment options to improve facture healing by utilizing or targeting MSC-macrophage crosstalk are also discussed.

Introduction

Bone fractures are one of the most common injuries seen in emergency departments, with nearly 4 million fractures seen in the United States in 2013 [1]. Despite the best treatment efforts, up to 10% of bone fracture cases still report undesirable outcomes; in the USA alone, 100,000 fractures per year result in painful non-union [2]. Treatment of these non-united fractures and bone defects constitutes a major health problem with significant clinical, social, and economic implications with an average cost of over 10,000 USD per non-union [3].

There are two major pathways for bone regeneration: intramembranous or endochondral ossification processes [[4], [5], [6], [7]]. Intramembranous ossification involves mesenchymal stem cells (MSCs) directly differentiating into osteoblasts which in turn deposit mineralized extracellular matrix. This type of healing is typically seen in rigidly fixed fractures with minimal fracture gap, and with fractures within the bone metaphysis. Fractures located in the diaphysis, with less mechanical stability, and a larger fracture gap heal via the classic stages of endochondral ossification: inflammation, soft then hard callus formation, and finally remodeling of the fracture site. In this mode of bone regeneration, the fracture hematoma is initially infiltrated by immune cells, mainly neutrophils and macrophages. Macrophages not only phagocytose necrotic cells and tissue debris at the fracture site but also initiate the recruitment of MSCs and vascular progenitor cells from the periosteum, bone marrow, and circulation [8,9]. As the inflammation subsides, MSCs and other progenitor cells proliferate, forming granulation tissue that ultimately forms cartilage callus to stabilize the fracture site [7,10]. In addition to providing mechanical stability cartilage functions as a scaffold for osteoblast-mediated bone deposition that allows for mineralization of the callus and closure of the fracture gap [11,12]. Osteoclasts then resorb immature woven bone and cartilage matrix, and with the subsequent deposition of mature lamellar bone, bone is restored to its pre-fracture structure and integrity [7,10].

The differentiation of MSCs and the subsequent formation of cartilage and bone at the fracture site is guided by several microenvironmental signals. These include growth factors released from the bone matrix as well as changes in the oxygen tension and mechanical microenvironment [7,10]. In particular, recent research has determined that both routes of fracture healing are based on carefully coordinated cross-talk between macrophages and bone forming cells.

MSCs, the precursor cells for bone and cartilage, were initially identified from human bone marrow with the ability to develop into fibroblastic colony-forming cells in vitro, and to regenerate heterotopic bone tissue in vivo [13,14]. The exact definition of the MSC remains controversial, but the term is generally used to describe a population of stem cells that resides in the peri-vascular niche of most tissues, and with the ability to differentiate into mesodermal tissues, such as bone and cartilage [15,16]. The International Society for Cellular Therapy has defined MSCs as cells that 1) adhere to plastic in vitro cell cultures; 2) have a certain surface marker profile (CD105+, CD73+, CD90+, and CD45−, CD34−, CD14−, CD11b−, CD79−, CD19−, and HLA-DR-); and 3) have the trilineage ability to differentiate into osteoblasts, adipocytes, and chondroblasts [17]. In addition to their ability to regenerate mesenchymal tissues, MSCs have wide immunomodulatory properties making them attractive targets for tissue engineering applications.

Macrophages are cells of innate immunity that are found in nearly all tissues, where they play a key role in maintaining normal tissue homeostasis [18]. During infection and inflammation, their numbers increase greatly via homing of regional and circulating monocyte precursors to the affected area [19]. During acute inflammation, macrophages contribute to the restoration of tissue homeostasis by phagocytosing invading micro-organisms, amplifying the inflammatory reaction, and recruiting additional immune cells [20]. As the tissue insult is cleared, macrophages contribute to tissue regeneration by secreting anti-inflammatory factors, recruiting progenitor cells, and producing growth factors that regulate the differentiation of these cells including angiogenesis [18,20]. This functional plasticity of macrophages has been conceptualized as macrophage polarization; inflammatory macrophages are called classically activated or M1 macrophages, while macrophages active in tissue regeneration are known as alternatively activated or M2 macrophages [21]. Importantly macrophages can switch from one mode of function to another, making them highly attractive targets for therapeutic interventions. In humans, there is a much wider spectrum of macrophage phenotypes, corresponding to relative differences in pro- and anti-inflammatory activities.

Macrophages are among the first cells to arrive to the fracture site, and have long been thought to contribute to the initial inflammation and debridement of the injury location (Fig. 1). Their key role also in the regulation of bone regeneration during both normal bone homeostasis and fracture healing has increasingly been appreciated. In addition to macrophages, closely related myeloid-lineage cells such as osteoclasts play complex roles in bone growth and regeneration [[22], [23], [24]], and delineating the exact roles that each of these myeloid lineage cell types play in bone regeneration remains challenging with currently available methods. Nevertheless the research has begun to identify some of the molecular mechanisms underlying the cross-talk between macrophages and bone forming cells and has led to new potential strategies to enhance bone regeneration by targeting the interaction between macrophages and MSCs.